Association of Anti-β 1 and Anti-M2 Antibodies with Autonomic Nervous System Modulation in Patients with Chronic Chagas Cardiomyopathy

Abstract Background: Different immune mechanisms of myocardial damage involved in the pathophysiology of Chagas disease coexist with high titers of autoantibodies induced by T. cruzi . There are few studies in the literature about the adaptive role of anti-β1 and anti-M2 antibodies in chronic Chagas cardiomyopathy (CCC). Objectives: To evaluate the association between anti-β1 and anti-M2 antibodies with heart rate variability (HRV) parameters on 24h Holter monitoring and the rate-pressure product (RPP) on cardiopulmonary exercise testing (CPET). Methods: Anti-β1 and anti-M2 antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) in 64 patients affected by CCC. Analysis of HRV was performed through the time-domain indices NNs, mean NN, SDNN, SDANN, SDNN index, NNNs, RMSSD, and pNN50. Spearman's correlation coefficient was used to assess the association between antibody titers and numerical variables. The Mann-Whitney test was used for comparison between two groups. Multiple linear regression was used to identify independent variables capable of explaining anti-β1 and anti-M2 antibody titers at the 5% significance level. Results: On 24h Holter, during the period of greatest parasympathetic activation (2:00-6:00 a.m.), an inverse association was found between anti-β1 titers and SDNN (rs=-0.13, p =0.041, n=43), as well as a direct association between anti-M2 titers and SDANN ( r s=0.317, p =0.039, n=43). Regarding CPET variables, anti-β1 titers were directly associated with RPP (rs=0.371, p =0.005, n=56). The subgroup of patients with a normal chronotropic response showed higher anti-β1 titers than the subgroup with an impaired response (p=0.023). RPP was an independent explanatory variable for anti-β1 titers, although with a low coefficient of determination (R2=0.147). Conclusion: The findings of this study suggest that, in patients with CCC, anti-β1 and anti-M2 antibodies may affect HRV parameters. RPP was directly associated with higher anti-β1 titers.

Impact of autoantibodies against the M2-muscarinic acetylcholine receptor on clinical outcomes in peripartum cardiomyopathy patients with standard treatment.

OBJECTIVES: To evaluate the impact of autoantibodies against the M2-muscarinic receptor (anti-M2-R) on the clinical outcomes of patients receiving the standard treatment for peripartum cardiomyopathy (PPCM). METHODS: A total of 107 PPCM patients who received standard heart failure (HF) treatment between January 1998 and June 2020 were enrolled in this study. According to anti-M2-R reactivity, they were classified into negative (n = 59) and positive (n = 48) groups, denoted as the anti-M2-R (-) and anti-M2-R (+) groups. Echocardiography, 6-min walk distance, serum digoxin concentration (SDC), and routine laboratory tests were performed regularly for 2 years. The all-cause mortality, cardiovascular mortality, and rehospitalisation rate for HF were compared between the two groups. RESULTS: A total of 103 patients were included in the final data analysis, with 46 in the anti-M2-R (+) group and 57 in the anti-M2-R (-) group. Heart rate was lower in the anti-M2-R (+) group than in the anti-M2-R (-) group at the baseline (102.7 ± 6.1 bpm vs. 96.0 ± 6.4 bpm, p < 0.001). The initial SDC was higher in the anti-M2-R (+) group than in the anti-M2-R (-) group with the same dosage of digoxin (1.25 ± 0.45 vs. 0.78 ± 0.24 ng/mL, p < 0.001). The dosages of metoprolol and digoxin were higher in the anti-M2-R (-) patients than in the anti-M2-R (+) patients (38.8 ± 4.6 mg b.i.d. vs. 27.8 ± 5.3 mg b.i.d., p < 0.0001, respectively, for metoprolol; 0.12 ± 0.02 mg/day vs. 0.08 ± 0.04 mg/day, p < 0.0001, respectively, for digoxin). Furthermore, there was a greater improvement in cardiac function in the anti-M2-R (-) patients than in the anti-M2-R (+) patients. Multivariate analysis identified negativity for anti-M2-R as the independent predictor for the improvement of cardiac function. Rehospitalisation for HF was lower in the anti-M2-R (-) group, but all-cause mortality and cardiovascular mortality were the same. CONCLUSIONS: There were no differences in all-cause mortality or cardiovascular mortality between the two groups. Rehospitalisation rate for HF decreased in the anti-M2-R (-) group. This difference may be related to the regulation of the autonomic nervous system by anti-M2-R.

Design, Synthesis and Primary Immunologic Evaluation of M2e-CRM197 Conjugate as a Universal Influenza Vaccine Candidate.

BACKGROUND: Influenza is a contagious respiratory illness caused by an acute infection of influenza viruses, among which influenza A virus causes seasonal epidemic infections nearly every year. Due to the unpredictability of the evolving influenza A virus and time-consuming vaccine development cycles, novel universal influenza vaccines designed to induce broadly cross-reactive immune responses against frequently mutant influenza A virus strains are urgently required. OBJECTIVE: The aim of this study was to synthesize a novel vaccine through the dual-site specific conjugation of the constant epitope of 23 amino acids (M2e) of the influenza A virus with a highly immunogenic carrier protein of Cross-Reacting Material (CRM197) under denaturation and to evaluate its primary immunogenicity in mice. METHODS: The antigen (M2e) and the carrier protein (CRM197) were linked with different types of hetero-functionalized linkers, α-Maleimide-ε-Hydrazide Polyethylene Glycol 2k (MAL-PEG-HZ) and N-ß-Maleimidopropionic Acid Hydrazide (BMPH) separately. The immunogenicity of the M2e-CRM197 conjugates with different types of linkers was evaluated in mice, and the M2especific total IgG and IgG-isotypes were determined by ELISA. RESULTS: Immunogenicity studies revealed that anti-M2e antibody could be induced by the conjugate products, M2e-PEG-CRM197 and M2e-BMPH-CRM197, by approximately 30 and 90-fold higher than that of the M2e group. In addition, the anti-M2e antibody level induced by M2e-PEG-CRM197 conjugate was three times higher than that of M2e-BMPH-CRM197 conjugate, and the former could simultaneously activate both cellular and humoral immune responses. CONCLUSION: The M2e-CRM197 conjugated vaccines we synthesized in this study are highly immunogenic compared with M2e alone. Besides, evidence presented here indicated that the hydrophilic, non-immunogenic and biocompatible chain of the cross-linker might be a better choice for the development of a conjugate vaccine.

Association of autoantibodies against the M2-muscarinic receptor with long-term outcomes in peripartum cardiomyopathy patients: A 5-year prospective study.

BACKGROUND: Peripartum cardiomyopathy (PPCM) is characterized by heart failure. Our previous study found that autoantibodies against the M2-muscarinic receptor (anti-M2-R) are increased in PPCM patients. We aimed to evaluate the association of anti-M2-R on prognosis of PPCM patients with standard treatment. METHODS: Synthetic peptides corresponding to the M2 receptor served as the target antigens in an enzyme-linked immunosorbent assay experiment. They were used to screen the sera of 80 PPCM patients, who were separated into anti-M2-R-negative and positive groups according to their anti-M2-R reactivity. Clinical assessment and echocardiography examination were performed at baseline and after 5 years with a standard treatment regimen. The endpoint events were compared after 5 years of follow-up. RESULTS: There were 76 PPCM patients who completed the final data analysis, including 36 in the anti-M2-R (+) group and 40 in the anti-M2-R (-) group. Both groups showed improvement in the left ventricular end-diastolic and end-systolic dimensions and the ejection fraction with standard treatment regimens for 5 years (all p<0.001). Patients in the anti-M2-R (-) group had greater tolerance and were more rapidly titrated to metoprolol, and they had better improvement in cardiac function than patients in the anti-M2-R (+) group (p<0.05). Patients in the anti-M2-R (-) group had a marked decrease in re-hospitalization (p<0.05), but not in all-cause mortality or cardiovascular mortality. Being positive for anti-M2-R increased the risk of PPCM (OR=4.7, 95% CI 1.8-12.2, p=0.002). CONCLUSIONS: PPCM patients, especially anti-M2-R (-) patients, have a relatively better prognosis than other patients. We posit that the presence of anti-M2-R may be involved in the pathogenesis of PPCM.

Association of ß1-Adrenergic, M2-Muscarinic Receptor Autoantibody with Occurrence and Development of Nonvalvular Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common sustained arrhythmia and is independently associated with increased risk of stroke and death. Although the exact mechanisms of AF are not completely elucidated, a large number of evidences demonstrate that autoimmunity may play an important role in the initiation, the progression, and the maintenance of AF. In this study, we aimed to compare anti-ß1-adrenergic receptor autoantibody (anti-ß1-R) and anti-M2-muscarinic receptor autoantibody (anti-M2-R) levels between nonvalvular AF patients and healthy control subjects. METHODS: The levels of serum anti-ß1-R, antinuclear antibodies, and anti-M2-R were measured in both groups by enzyme-linked immunosorbent assay (ELISA). High-sensitivity C-reactive protein (hs-CRP) and interleukin (IL)-6 concentration were measured, respectively, by immunoturbidimetry and chemiluminescence. RESULTS: Anti-ß1-R and anti-M2-R levels were significantly higher in patients with nonvalvular AF than in healthy controls (anti-ß1-R 221.11 [132.38-291.69] ng/mL vs 198.14 [125.70-278.40] ng/mL, P < 0.01; anti-M2-R 271.81 [144.99-378.20] ng/mL vs 235.01 [121.53-358.99] ng/mL, P < 0.01). Compared with the control group, the serum levels of IL-6 and hs-CRP were higher in the nonvalvular AF group (IL-6 19.65 ± 5.6 pg/mL vs 6.79 ± 1.09 pg/mL, hs-CRP 6.03 ± 1.35 mg/L vs 2.73 ± 0.63 mg/L, P < 0.05). Antinuclear antibody (ANA) levels were similar between two groups (ANA 10.55 [1.86-271.8] U/mL vs 10.49 [1.303-161.7] U/mL, P > 0.05). The baseline value of serum anti-ß1-R (odds ratio [OR]: 13.176, P < 0.001), anti-M2-R (OR: 4.41, P < 0.001), IL-6 (OR: 6.126, P < 0.05) levels, and left atrial diameter (OR: 5.781, P < 0.05) were independent predictors of nonvalvular AF by multivariable analyses. CONCLUSION: We found a significant association between circulating serum anti-ß1-R, anti-M2-R, IL-6 levels, and nonvalvular AF. We presume that the autoimmunity and inflammation might take part in electrical remodeling and structural remodeling of left atrium.

Efficacy and safety of treatment with an anti-m2e monoclonal antibody in experimental human influenza.

BACKGROUND: The efficacy of TCN-032, a human monoclonal antibody targeting a conserved epitope on M2e, was explored in experimental human influenza. METHODS: Healthy volunteers were inoculated with influenza A/Wisconsin/67/2005 (H3N2) and received a single dose of the study drug, TCN-032, or placebo 24 hours later. Subjects were monitored for symptoms, viral shedding, and safety, including cytokine measurements. Oseltamivir was administered 7 days after inoculation. RESULTS: Although the primary objective of reducing the proportion of subjects developing any grade ≥2 influenza symptom or pyrexia, was not achieved, TCN-032-treated subjects showed 35% reduction (P = .047) in median total symptom area under the curve (days 1-7) and 2.2 log reduction in median viral load area under the curve (days 2-7) by quantitative polymerase chain reaction (P = .09) compared with placebo-treated subjects. TCN-032 was safe and well tolerated with no additional safety signals after administration of oseltamivir. Serum cytokine levels (interferon γ, tumor necrosis factor α, and interleukin 8 and 10) were similar in both groups. Genotypic and phenotypic analyses showed no difference between virus derived from subjects after TCN-032 treatment and parental strain. CONCLUSIONS: These data indicate that TCN-032 may provide immediate immunity and therapeutic benefit in influenza A infection, with no apparent emergence of resistant virus. TCN-032 was safe with no evidence of immune exacerbation based on serum cytokine expression. Clinicaltrials.gov registry number. NCT01719874.

A case of conventional antimitochondrial antibody test-negative primary biliary cirrhosis / 대한내과학회지

Primary biliary cirrhosis is a chronic progressive cholestatic liver disease of unknown cause that usually affects middle-aged women and eventually leads to cirrhosis and liver failure. It is characterized by the progressive destruction of small intrahepatic bile ducts, portal inflammation, and progressive scarring. The diagnosis is made by these characteristic pathologic findings and the presence of antimitochondrial antibody. Immunofluorescence, the most widely used method for determining antimitochondrial antibody, is less sensitive and specific than ELISA or immunoblotting and influenced by observer interpretation. Therefore, it is important to detect anti-M2 antibody, the most specific antibody of primary biliary cirrhosis, by ELISA or immunoblotting when antimitochondrial antibody is not detected by immunofluorescence method which can lead to the incorrect diagnosis as autoimmune cholangitis. We describe a case of primary biliary cirrhosis with antimitochondrial antibody negative by immunofluorescence, anti-M2 antibody positive by ELISA. We confirmed primary biliary cirrhosis by liver biopsy.

Polymorphisms of PPARgamma2 gene in patients with type 2 diabetes mellitus and obesity / 대한내과학회지

BACKGROUND: Peroxisome proliferator activated receptor-gamma (PPAR-gamma) is a nuclear receptor that regulate adipocyte differentiation and modulate intracellular insulin-signaling events. As such, PPARgamma is a candidate gene for several human disorders including obesity and type 2 diabetes mellitus. The objective of our study was to examine the relationship between genetic variation of PPARgamma2 and diabetes and obesity in Korean subjects. METHODS: We studied 99 subjects with type 2 diabetes mellitus, 128 obesity patients and 97 controls. Screening for mutation at codon 12 and 115 of PPARgamma2 were carried out by PCR-RFLP analyses. Statistical significance was evaluated by Chi-square test. RESULTS: The allele frequency of the Pro12Ala PPARgamma2 variant were 0.05 in controls, 0.06 in type 2 diabetes group, and 0.07 in obesity group (p=0.47). Pro115Gln variant were only proline homozygote in all groups. Genotype frequencies were also similar and conformed to expectations of the Hardy-Weinberg rule. The presence of PPARgamma2 gene variant was no associated with concentrations of total cholesterol, triglyceride, HDL-cholesterol, and also with fasting glucose. CONCLUSION: We concluded that the Pro12Ala and Pro115Gln PPARgamma2 missense mutation may not be associated with type 2 diabetes mellitus and obesity in Korean patients.